Tenomodulin (Tnmd) is a tenocyte biomarker with three isoforms. Results from a knockout mouse model of isoforms I and II, as well as RNAi experiments that knocked down all three isoforms, reduced cell proliferation. Strain in vitro localized Tnmd immunochemically to the nucleus. Given a role in cell proliferation and a nuclear involvement, we hypothesized that Tnmd might have a role in cell cycle regulation. Prometaphase cells from tenocytes or HeLa cells showed robust Tnmd staining throughout the cytoplasm. Cells released at 16h from the nocodazole block showed Tnmd localization in a peri-chromatin boundary in prometaphase. One h post-block, cells in metaphase showed robust Tnmd staining in a punctate border at the periphery of the chromosomes and in the cytoplasm. At two h post-block, at anaphase, Tnmd localization was peri-chromosomal at the segregating chromosomes and in dual puncta at poles in the cell where centrioles locate. At 2.5h post-block, at telophase, Tnmd signal was less robust, still in a peri-chromosomal localization with polar puncta. GFP isoforms one and two were cytoplasmic, showing globules of GFP-Tnmd, with a focus around the nuclear envelope. GFP-isoform I had a clear overlap with anti-Tnmd antibody signal as did isoform II. GFP-isoform I was cytoplasmic at prometaphase and in a distinct halo around the chromatin, then tracked with the segregating chromosomes throughout mitosis. This observation may place Tnmd isoform I at the checkpoint complex in anaphase as a novel but dispensable regulatory protein in the cell cycle, since RNAi results demonstrated reduced cell proliferation.